My research objective is to elucidate the mechanism by which vitamin B12 transport proteins facilitate the cellular uptake of vitamin B12. Plasma transcobalamin II facilitates the uptake of vitamin B12 by a number of different cells. We have isolated human and rabbit transcobalamin II in homogeneous form and have developed methods for labeling these proteins with radioactive iodine. These labeled proteins will be used to study the role and fate of trancobalamin II during the complex process in which vitamin B12 binds to cell surfaces, enters cells, migrates to various sub-cellular fractions, and exits from cells. 58Co-vitamin B12 and a variety of 57Co-vitamin B12 analogues will be employed to study the importance of, and alterations which occur in, the structure of vitamin B12 during these processes. Human R-type vitamin B12-binding proteins with low sialic acid contents transport vitamin B12 exclusively to the liver. 125I-labeled R-type proteins, 58Co-vitamin B12, and 57Co-vitamin B12 analogues will be employed to elucidate this process and to determine its biological significance. We also plan to characterize the process by which structurally abnormal R-type proteins are synthesized in chronic myelogenous leukemia and other myeloproliferative diseases. Intrinsic factor facilitates the uptake of vitamin B12 by ileal mucosal cells. We plan to isolate and characterize the ileal cell surface receptor for the intrinsic factor-vitamin B12 complex. We plan to label intrinsic factor in vivo with 3H-amino acids and utilize this radioactive protein to elucidate the mechanism by which intrinsic factor facilitates the ileal absorption of vitamin B12.